Due to their physiological relevance, variety and ubiquitousness, transferases, especially kinases, have become one of the most important and widely studied families of enzymes in biochemical and medical research. Studies have shown that protein and lipid kinases are key regulators of many cell functions, including signal transduction, transcriptional regulation, cell motility, cell division and cellular responses to drugs, toxins, and pathogens.
Protein kinases play crucial roles in the modulation of a wide variety of cellular events. These enzymes act by transferring phosphate residues to certain amino acids in intracellular polypeptides to bring about the activation of these protein substrates and set in motion a cascade of activation controlling events including growth, differentiation and division of cells. Protein kinases have been extensively studied in the field of tumour biology. A lack of controlled activity of kinases in cells is believed to lead to the formation of tumours. The pharmaceutical industry is constantly in search of drugs that target these kinases to help with the treatment of a wide variety of tumours. There are over 500 protein kinases (about 2 to 2.5% of the human genome) that are involved in the regulation of cell functions. They occur as both transmembrane and cytosolic enzymes, and they phosphorylate serine, threonine and tyrosine amino acid residues. Based on these substrate specificities, the kinases are divided into two groups, the serine/threonine kinases and tyrosine kinases.
Serine/threonine kinases include cyclic AMP and cyclic GMP dependent protein kinases, calcium and phospholipid dependent protein kinase, calcium and calmodulin-dependent protein kinases, casein kinases, cell cycle protein kinases and others. These kinases are usually cytoplasmic or associated with the particulate fractions of cells, possibly by anchoring proteins.
Tyrosine kinases phosphorylate tyrosine residues. These particular kinases are present in much smaller numbers but play an equally important role in cell regulation. These kinases include several soluble enzymes such as the src family of protein kinases and receptors for growth factors such as epidermal growth factor receptor, insulin receptor, platelet derived growth factor receptor, and others. Studies have indicated that many tyrosine kinases are transmembrane proteins with their receptor domains located on the outside of the cell and their kinase domains on the inside.
Lipid kinases also play important roles in the intracellular signal transduction and have been grouped into four major classes. Exemplary lipid kinases include PI3 kinases and phosphatidylinositol 4-kinases.
Sugar kinases and other phosphotransferases also play a major role in cellular metabolism, proliferation and apoptosis.
With phosphorylation events involved in so many cell functions and diseases, identifying kinase activity is tremendously important. Current types of assays used to measure kinase activity include Fluorescence Resonance Energy Transfer (FRET) assays, Fluorescent Polarization (FP) assays, and assays based on radioactivity such as Scintillation Proximity Assay (SPA). FRET assays used to detect kinase activity utilize a protein or peptide substrate that is linked to a fluorescent molecule and another fluorescently labeled probe. The two fluorescent molecules are in close proximity only when the substrate is phosphorylated and recognized by the labeled probe. Thus, when phosphorylated by a kinase, the energy of the label is passed to the fluorescent molecule (the acceptor) through resonance. The ability of a higher energy donor fluorophore to transfer energy directly to a lower energy acceptor molecule causes sensitized fluorescence of the acceptor molecule and simultaneously quenches the donor fluorescence. In this case, the fluorescence of the donor is “quenched” by the proximity to the acceptor and the energy of the donor is transferred to the acceptor in a non-radioactive manner. The efficiency of energy transfer is dependent on the distance between the donor and acceptor chromophores according to the Forster equation. In most cases, no FRET is observed at distances of greater than 100 angstroms, and thus the presence of FRET is a good indicator of close proximity. Accordingly, in a FRET assay to detect kinase activity that employs a protein or peptide substrate that is linked to a fluorescent molecule and another fluorescently labeled probe, if the kinase is inhibited, the two fluorescent molecules remain separated and no FRET occurs.
FRET based assays have a number of drawbacks including a large number of false hits, e.g., due to fluorescent interference with the compounds being tested, a narrow dynamic range, and performance issues associated with the antibody employed in the assay.
FP assays are based on binding of a high affinity binding reagent, such as an antibody, a chelating atom, or the like, to a fluorescent labeled molecule. For example, an antibody that binds to a phosphorylated fluorescent labeled peptide but not a non-phosphorylated fluorescent labeled peptide can be used for a kinase assay. Other methods utilize a fluorescent labeled antibody that binds ADP, the other kinase reaction product, in monitoring kinase activity. When the fluorescent label is excited with plane polarized light, it emits light in the same polarized plane as long as the fluorescent label remains stationary throughout the excited state (duration of the excited state varies with fluorophore, and is 4 nanoseconds for fluoroscein). If polarized light is used to excite the fluorophore, the emission light intensity can be monitored in both the plane parallel to the plane of polarization (the excitation plane) and in the plane perpendicular to the plane of polarization. An FP assay requires a high affinity binding reagent, e.g., an antibody, capable of binding with high specificity to the fluorescent labeled molecule. The time consuming and costly optimization of an antibody binding with specific fluorescent labeled molecules such as peptides is required where antibodies are used. Additionally, in the FP assay there is the potential for phosphorylated protein and other reaction components, e.g., lipids and detergents, to interfere with the polarization.
Kinase assays that use radioactive labels include SPA. In SPA, modified ligand-specific or ligand-capturing molecules are coupled to fluoromicrospheres, which are solid-phase support particles or beads impregnated with substances that emit energy when excited by radioactively labeled molecules. When added to a modified ligand such as radio-labeled phosphopeptide in a mixture with nonphosphorylated peptide, only the phosphopeptide is captured on a fluoromicrosphere, bringing any bound radiolabeled peptide close enough to allow the radiation energy emitted to activate the fluoromicrosphere and emit light energy. If the concentration of fluoromicrospheres is optimized, only the signal from the radio-labeled ligand bound to the target is detected, eliminating the need for any separation of bound and free ligand. The level of light energy emitted may be measured in a liquid scintillation counter and is indicative of the extent to which the ligand is bound to the target. A SPA requires the fluoromicrospheres to settle by gravity or be centrifuged, adding an additional step and time to the assay. In addition, due to the high energy of 32P used in this assay, most of the radioactivity passes through without being captured by the fluoromicrospheres.
Other methods also have been developed for detecting kinase activity that are based on luminescence detection, either by bioluminescence or chemiluminescences. Generally, these methods rely on specific substrates and antibodies, the use of microchips and fluorescent label probes, substrate concentration in a sample, the use of multiple steps and reagents (U.S. Pat. No. 6,599,711) or are limited to specific kinases (Sala-Newby et al., 1992).
Many currently available kinase assays perform well with enzymes that consume a high amount of ATP during the reaction, but are not sensitive enough to detect the small changes in ATP amount that define kinases with low ATP turnover, like growth factor receptor kinases. On the contrary, the available assays that could analyze this type of enzyme are specific for a subset of kinases and/or not sensitive enough to be used with a broad range of kinases.